Oocyte/DNA microinjection and oviduct transfer
Six-week old F1 female mice (B6D2F1) were obtained from mating of
C57BL/6 and DBA2. Mice were superovulated with 10IU of pregnant mare’s
serum gonadotropin (Ningbo Hormone Products CO., Ltd, Ningbo,
Zhengjiang, China) and followed by 5IU of human chorionic gonadotropin
(Ningbo Hormone Products CO., Ltd, Ningbo, Zhengjiang, China) 48hrs
later. Superovulated B6D2F1 females were crossed with B6D2F1 males.
Fertilized eggs at pronucleus stage were collected in M2 medium.
Mixtures of pTyr-gRNAs (2.35ng/ul) and BE4 plasmids (2.64ng/ul) were
injected into nucleus in a droplet of M2 medium using inverted
microscope equipped with a pair of micromanipulators (Olympus, Tokyo,
Japan). Then the injected embryos were incubated in M16 culture medium
at 37 °C, 6% CO2 overnight, followed by transfer into
the oviduct of a recipient mother at two-cell stage.